Structural Basis for Antiarrhythmic Drug Action Abstract Voltage-gated sodium (Na) channels initiate action potentials in cardiac myocytes, and they are the molecular targets for Class I antiarrhythmic drugs (AADs) used in the control of life- threatening cardiac arrhythmias. The structural basis for antiarrhythmic drug action is unknown. Na channels are large integral membrane proteins with 24 transmembrane segments. We have recently determined the crystal structure of an ancestral bacterial Na channel (NavAb) at 2.7 A resolution in a pre-open state with voltage sensors activated but the pore closed. This remarkable structure defines the structural basis for voltage sensing, pore opening and closing, and ion selectivity. Moreover, this structure reveals an entirely unexpected feature- fenestrations that lead laterally from the lipid bilayer into the pore and potentially provide an access pathway for entry of hydrophobic pore-blocking AADs to their receptor site in closed Na channels. The receptor site for antiarrhythmic drugs is located within the lumen of the pore. AADs block Na channels in rapidly firing cells more effectively because they reach their receptor site more rapidly when the pore is open. These drugs also block Na channels more effectively in damaged, depolarized cardiac myocytes because they bind with highest affinity to the inactivated state of the channel that is preferred at depolarized membrane potentials. Three different groups of Class I AADs (Ia, Ib, and Ic) modify Na channel function differentially and are useful in treatment of distinct classes of arrhythmias. Availability of the first high-resolution N channel structure now allows us to determine the structural basis for the complex blocking mechanism, use-dependence, and subclass specificity of AADs, which are essential for their clinical use. We will determine the structure of NavAb in the inactivated state, which has highest affinity for AADs. We will determine the structure of the NavAb channel with an antiarrhythmic drug bound by x-ray crystallography and define the structural basis for state-dependent drug binding. In addition, we will construct a human AAD receptor site in NavAb, determine the structure of the drug-bound, humanized Na channel, and define the structural basis for subclass-selective actions of Class Ia, Ib, and Ic AADs. These results will open a new era of Na channel pharmacology by revealing the structural basis for the state-dependent drug binding and block of this ion channel. Our results will be crucial in illuminating the structural determinants of high-affinity binding, the underlying mechanism for the complex, state- dependent access pathway of drugs to the AAD receptor site, and the structural basis for the selective binding and action of the Class Ia, Ib, and Ic AADs. This information will provide the structural basis for discovery and development of safer and more effective AADs.